Within the cell, transcriptional selectivity of eukaryotic genes is mediated by complex control regions composed of different combinations of promoter and enhancer elements. These regions are arrayed in tandem to allow multiple distinct regulatory factors to function coordinately to potentiate RNA synthesis. This mosaic arrangement of eukaryotic transcriptional regulatory elements provides different genes with the possibility of utilizing some of the same regulatory elements.
Enhancers are sequence-specific DNA transcriptional regulatory elements that function in cis to stimulate the transcription of genes placed in proximity to them. Generally, elements that function in cis are recognition sites for cellular proteins (Dynan, W. S. et al., Nature 316:774-778 (1985)). The cellular proteins which recognize enhancer sequences are often expressed in a manner which is tissue-specific or species-specific, or dependent upon the hormonal environment. Upon binding of the appropriate protein to the enhancer region, transcription of genes under the control of, that is, operably-linked to the enhancer is facilitated, resulting in an increased transcriptional expression of the gene, and thus in an increased expression of any protein for which the gene codes.
Enhancers are not orientation dependent elements like promoter regions are. Enhancer sequences can be oriented in either direction relative to the direction of transcription of the operably-linked gene. In addition, the sequence itself may be located anywhere in the general area of the gene, such as 5' to the promoter region, 3' to the transcriptional termination site, or even within a transcribed region of the gene, for example, in an intron. A gene may be under the transcriptional regulatory influence of multiple copies of the same enhancer, or the gene may be under the transcriptional regulatory influence of a group of different enhancers, each enhancer in the group conferring a different regulatory response on the operably-linked gene. Examples of these responses include an ability to transcriptionally respond to different agents or hormones, and tissue-specific expression of the gene.
Because of their relative orientation independence, enhancers can be located at varying distances from the promoter and transcription unit of the gene and yet still be operably-linked to that gene. The transcription unit is that sequence of a gene which is transcribed. The distance will vary with the transcriptional strength of the promoter and enhancer. Typically, on the average, enhancers are located within 200 bases upstream from the promoter site which itself determines the base at which transcription begins.
Cyclic adenosine monophosphate (cAMP) is the intracellular second messenger for many hormones or biological mediators and is known to be active in the regulation of gene expression in both prokaryotes and eukaryotes. In eukaryotes, the regulation of transcription by cAMP has been extensively studied in animals and tissue culture cells. Increasing the intracellular cAMP concentration with hormones such as glucagon or other agents such as cAMP analogs or beta-adrenergic agonists induces the transcription of many genes in a tissue-specific manner, including somatostatin (Montminy, M. R. et al., Proc. Natl. Acad. Sci. USA 83:6682 (1986)), the alpha subunit of human chorionic gonadotropin (Silver, B. J. et al., Proc. Natl. Acad. Sci. USA 84:2198 (1987); Jameson, J. L. et al., Endocrinology 119:2570 (1986); Delegeane, A. M. et al., Mol. Cell. Biol. 7:3994 (1987); Jameson, J. L. et al., Mol. and Cell. Biol. 7:3032 (1987); Deutsch, P. J. et al., Bio. Chem. 262:12169 (1987)); phosphoenolpyruvate carboxykinase (Short, J. M. et al., Biol. Chem. 261:9721-9726 (1986)), tyrosine hydroxylase (Lewis, E. J. et al., Proc. Natl. Acad. Sci. USA 84:3550-3554 (1987)), and c-fos (Greenberg, M. E. et al., J. Biol. Chem. 160:14101-14110 (1985)).
Cyclic AMP-responsive genes contain a sequence homologous to the sequence TGACGTCA located on the 5' side of their mRNA cap sites. This sequence has been termed a cAMP-responsive enhancer element (CRE). Deletion mutagenesis of cAMP-inducible genes has shown that the cAMP-responsive enhancer element is contained within a domain necessary for cAMP-mediated induction of transcription.
Similar consensus DNA regulatory elements involved in the stimulation of gene transcription have been identified for other molecules, such as for the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) (Imbra, R. J. et al., Mol. and Cell. Bio. 7:1358 (1987); Angel, P. et al., Cell 49:729 (1987); Tsukada, T. et al., Bio. Chem. 262:8743 (1987); Angel, P. et al., Mol. and Cell. Biol. 6:1760 (1986); Chiu, R. et al., Nature 329:648 (1987); Angel, P. et al., Mol. and Cell. Biol. 74:2256 (1987); Comb, M. et al., Nature 323:353 (1986)). However, notably, the sequence of the octameric cAMP-response element, CRE, (5'-TGACGTCA-3') differs from that of the heptameric TPA-response element, TRE, (5'-TGAGTCA-3') by a single base.
Early studies suggested that transcriptional stimulation by both cAMP and TPA was mediated through a common DNA sequence present in the 5' regulatory region of the enkephalin gene, 5'-TGCGTCA-3' (Comb, M. et al., Nature 323:353 (1986)). However, a DNA binding protein of 47 Kd (AP-l or c-jun) was isolated and shown to mediate TPA but not cAMP induction of SV40 gene transcription through a mechanism involving sequence-specific binding to the TRE motif (Lee, W. et al., Cell 49:741 (1987)). Similarly, a 43 Kd protein termed CRE-binding protein (CREB) has been identified that binds to a CRE sequence in the 5' regulatory region of the rat somatostatin gene (Montminy, M. R. et al., Nature 328:175 (1987)). In placental JEG-3 cells, a 38 Kd protein was shown to bind to CRE (Deutsch, P. J., et al., Proc. Natl. Acad. Sci. USA 85:7922 (1988)). However, the sequence of CREB had not previously been determined, precluding the undertaking of detailed structural or functional studies.
Anti-sense RNA refers to RNA synthesized with a sequence complementary to that found in a specific mRNA. Anti-sense RNA has been used to inhibit, in a specific manner, the expression of the protein whose mRNA is being hybridized by the anti-sense RNA. Inhibition by hybridization in eukaryotes is thought to occur at the level of processing of the mRNA (thus preventing its translocation to the cytoplasm) while in prokaryotes it is thought to occur at translation of the mRNA. At either step, the ultimate result is to effectively stop expression of the target protein whether the system is bacteria, plants or other eukaryotic systems (Knecht, D. A. et al., Science 236:1081-1086 (1987); Van Der Krol, A. R. et al., Nature 333:866-869 (1988); Cabrera, C. V. et al., Cell 50:659-663 (1987); Boulay, J. L. et al., Nature 330:395-398 (1987); Rothstein, S. J. et al., Proc. Natl. Acad. Sci. USA 84:8439-8443 (1987); Ecker, J. R. et al., Proc. Natl. Acad. Sci. USA 83:5372-5376 (1986); Lichtenstein, D., Nature 333:801-802 (1988)). However, it has not previously been known to use CAMP with anti-sense RNA technology to control the expression of specific proteins in a manner capable of acute regulation in response to the levels of cAMP in the system.